Purification, cloning, and expression of a cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi
Purification, cloning, and expression of a cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi
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DOI：
10.1074/jbc.271.26.15373
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发表时间：
1996-06-28
影响因子：
4.8
通讯作者：
Gibson, BW
中科院分区：
文献类型：
--
作者：
Tullius, MV;Munson, RS;Gibson, BW
从杜克雷嗜血杆菌菌株35000细胞裂解物中分离N-乙酰神经氨酸胞苷酰转移酶（EC 2.7.7.43）（CMP-NeuAc合成酶）并进行部分表征。该酶催化CTP和NeuAc反应形成CMP-NeuAc，其是唾液酸转移酶使用的核苷酸糖供体。以往的研究表明，H。ducreyi含有连接到N-乙酰乳糖胺的末端唾液酸，并且这种修饰可能对其发病机制很重要。因此，研究唾液酸在A.在杜克雷氏菌致病机理中，使用衍生自NH 2-末端序列数据的简并寡核苷酸探针克隆编码CMP-NeuAc合成酶的基因，并确定核苷酸序列。CMP-NeuAc合成酶基因的衍生氨基酸序列与其他CMP-NeuAc合成酶具有同源性，并且在较小程度上与CMP-2-酮基-3-脱氧-D-甘露-辛酮糖酸合成酶具有同源性。将该基因克隆到T7表达载体中，在大肠杆菌中表达蛋白质，并通过阴离子交换、绿色19染料和疏水相互作用色谱法纯化至表观均一性。最后一步产生20 mg纯蛋白质/升培养物，该蛋白质的预测分子量为25440.6 Pa，这通过电喷雾质谱法证实（M（expt）= 25439.9 +/- 1.4 Pa）。通过尺寸排阻色谱法，该酶似乎以二聚体形式存在。与其他细菌CMP-NeuAc合成酶相比，H. ducreyi酶表现出不同的底物特异性，也能够使用N-羟乙酰神经氨酸作为底物。
An N-acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) was isolated from a Haemophilus ducreyi strain 35000 cell lysate and partially characterized. The enzyme catalyzes the reaction of CTP and NeuAc to form CMP-NeuAc, which is the nucleotide sugar donor used by sialyltransferases. Previous studies have shown that the outer membrane lipooligosaccharides of H. ducreyi contain terminal sialic acid attached to N-acetyllactosamine and that this modification is likely important to its pathogenesis. Therefore, to investigate the role of sialic acid in a. ducreyi pathogenesis, the gene encoding the CMP-NeuAc synthetase was cloned using degenerate oligonucleotide probes derived from NH2-terminal sequence data, and the nucleotide sequence was determined. The derived amino acid sequence of the CMP-NeuAc synthetase gene has homology to other CMP-NeuAc synthetases and to a lesser extent to CMP-2-keto-3-deoxy-D-manno-octulosonic acid synthetases, The gene was cloned into a T7 expression vector, the protein expressed in Escherichia coli, and purified to apparent homogeneity by anion exchange, Green 19 dye, and hydrophobic interaction chromatography. The final step yielded 20 mg of pure protein/liter of culture, The protein has a predicted molecular mass of 25440.6 Pa, which was confirmed by electrospray mass spectrometry (M(expt) = 25439.9 +/- 1.4 Pa). The enzyme appears to exist as a dimer by size exclusion chromatography. In contrast to other bacterial CMP-NeuAc synthetases, the H. ducreyi enzyme exhibited a different substrate specificity, being capable of also using N-glycolylneuraminic acid as a substrate.